A Plasmonic Nanoledge Array Sensor for Selective Detection of Cardiovascular Disease Biomarkers in Human Whole Blood

Optical sensors face challenges when detecting ultralow amounts of analytes in whole blood, including signal quenching due to optical absorption and false positives due to nonspecific binding. This study introduces gold nanoscale array features termed nanoledges (NLs), which interact with incident white light to produce a transmitted surface plasmon resonance (tSPR) signal. This extraordinary optical transmission (EOT) spectrum occurs in the near-infrared (NIR) region, thereby minimizing signal quenching caused by visible-light absorption from blood proteins and pigments. To develop a sensitive, selective, and label-free optical biosensor for detecting various levels of cardiac troponin I (cTnI) in very small volumes of whole blood samples, DNA aptamers are tethered to the NL surface, specifically binding to the cTnI biomarker. This biological binding activity alters the refractive index at the NL surface, causing a peak shift in the EOT spectrum and enabling quantification of cTnI levels. The NL array chip demonstrated high sensitivity for cTnI detection in buffer, human serum (HS), and human whole blood (HB), with detection limits of 0.079, 0.084, and 0.097 ng/mL, respectively. Control measurements using blank target mediums and those containing up to 125 ng/mL of other proteins, such as myoglobin, creatine kinase, and heparin, showed minimal interference and high specificity. The NL plasmonic array’s performance in biosensing underscores its promise for clinical analysis and its potential development as a point-of-care platform for early cardiovascular disease (CVD) diagnostics.


Fig. S1.
EOT due to solvents at different refractive indexes for sensitivity evalluation.

Fig. S2.
EOT due to SAM formations and aptamer immobilization.

Fig. S3.
EOT recorded upon cTnI incubation with aptasensor at different binding times.

Fig. S4.
Graphical fit of concentration dependent wavelength peak position.

Fig. S5.
Dependence of adlayer film thickness on the change in wavelength position and cTnI concentration.

Fig. S6.
Representative plots of Raw and smoothed EOT spectral curves.

Fig. S7.
Enlarged EOT peak spectra for cTnI measurement in (A) Whole blood sample (B) Human serum (C) PBS buffer.

Fig. S8.
EOT spectra showing selectivity and reproducibility of NL sensors.

Fig. S9.
Stability of Aptasensor measured EOT spectra at three days interval for twenty-one days.
Table S1.Adlayer thickness as a function of concentration and wavelength peak shift.
The principle of the sensing scheme is based on the extraordinary optical transmission (EOT) of light through the NL array.According to Bethe's theory, the diffraction limit of light restrict light to pass through an orifice smaller than its wavelength. 1 However, subwavelength apertures in metal films can transmit light due to surface plasmons excitation which mediates light tunnelling and transmission, and hence giving birth to the phenomenon of EOT. 2,3The wavelength of optical transmission through the NL aperture can be approximated by Eq. 1. [4][5]   = From Eq. 1, the spectral characteristics of the EOT depends on the effective refractive index (  ) at the metal/dielectric interface, the hight (h), width (W) and periodicity (P) of the nanoaperture and the resonance wavelength (λ) at the phase matching condition.Since the size, shape and geometry of the metal aperture determines the efficiency of SPP excitation, the SPR mediated EOT is also dependent on the geometry of the nanoaperture.The wavelength sensitivity of EOT to changes in RI at the metal surface is what informed our decision to use the NL EOT-based system to interrogate biological binding interactions at the metals surface.Note that the inner ledges are designed to enable SPR excitation for EOT and to capture the biomarkers and thus provide increased sensitivity to the device. 6Initially, clean gold nanoledge is chemically modified to have a terminal carbonyl group that covalently bind with an amine modified cTnI aptamer. 7Since the aptamer is designed to specifically target cTnI protein, incubating the aptamer modified sensor with cTnI results in another binding interaction.
Following the detection of different concentrations of cTnI in whole blood, human serum, and PBS buffer, we established a linear regression equation, given by λ(nm) = 2.64 ± 0.085 x log C ( ng mL ) + 852.75 (nm) with  2 = 0.982, λ(nm) = 2.14±0.09x log C (ng/mL) + 844.47 (nm), with R 2 value of 0.993, and λ(nm) = 2.2±0.16x log C (ng/mL) + 833.3 (nm), with R 2 = 0.980, respectively (Figure 3).The Binding Event Analysis.The red shift in resonance EOT wavelength resulting from the increase in cTnI concentration (Figure 2) is taken to be due to the formation of a thin organic layer bound to the metal's surface.The thickness of the monolayer at different stages of the multi-layer formation is estimated based on Eq. 2. 8 The change in SPR response, ∆, is defined as the shift in wavelength of the resonance peak in The best fit was obtained using ld=123 nm.

Figure S2 .
Figure S2.EOT due to SAM formations and aptamer immobilization.

Figure S3 .
Figure S3.EOT upon TnI incubation with aptasensor at different binding times.This was obtained by transferring 50 μL of 0.156 ng/mL cTnI sample in PBS on to a clean aptamermodified sensor chip.

8 𝑑Figure S4 .
Figure S4.Graph of tSPR versus concentration (in PBS) showing experimental data points (black dots) fitted with the equation,

Figure S8 .
Figure S8.Normalized transmitted spectra of the NL sensor chip with (A) myoglobin, (B)

Figure S9 .
Figure S9.Stability of Aptasensor measured EOT spectra at 3 days interval for twenty-one days using 10 ng/mL TnI in PBS.

Table S1 .
Adlayer thickness as a function of concentration and wavelength peak shift (ld=123 nm)